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Western Blot Protocol

Sample protein preparation

Monolayer Cells

Grow cells to 80-90% confluency in a 100 mm dish, remove culture medium and rinse cell monolayer with 1x PBS at room temperature.

Add 0.3-0.5 ml lysis buffer enriched with protease inhibitor cocktail to the monolayer cells in the plate. Scrape the cells off and transfer the lysate to an eppendorf tube. Incubate for 30 minutes on ice.

Centrifuge cell lysate at 14000 rpm for 15 minutes at 4°C. The supernatant fluid is the total cell lysate. Transfer the supernatant to a new eppendorf tube. This is your whole cell lysate.

Tissue Samples

Weigh tissue and cut into very small pieces. Frozen tissue should be sliced very thinly and thawed in lysis buffer containing proteinase inhibitors.

Sonicate the tissue, and incubate on ice for 30 minutes. Transfer to eppendorf tubes, centrifuge at 14000 rpm for 15 minutes at 4°C. The supernatant fluid is the total cell lysate.

Make protein concentration assay for each sample.

Electrophoresis and blotting

a. Add an appropriate amount of loading buffer to each sample. Vertex the tubes.
b. Boil at water bath for 5 minutes.
c. Load 5-100 ug total protein in a volume that is appropriate for the size of the wells. Load 10 ul protein marker to 1-2 blank lanes
d. Electrophorese proteins for the appropriate time and voltage according to the manufacturer’s instruction.
e. Transfer proteins from the gel to a suitable membrane (e.g. nitrocellulose, PVDF, etc.) following standard method.

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